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Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR

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Abstract

Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716–730, 2010; J Mol Biol 426:1109–1127, 2014; J Biol Chem 291:13098–13112, 2016; J Am Chem Soc 138:8538–8546, 2016; J Am Chem Soc 138:12029–12032, 2016; J Am Chem Soc 134:6455–6466, 2012; J Am Chem Soc 132:1976–1987, 2010; J Am Chem Soc 135:17793–17803, 2013; Proc Natl Acad Sci USA 112:14617–14622, 2015; J Am Chem Soc 138:14066–14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements. Nonetheless, the relatively high molecular weight of HIV-1 CA leads to congestion of solid state NMR spectra of fully isotopically labeled assemblies that has been an impediment to further progress. Here we describe an efficient protocol for production of segmentally labeled HIV-1 CA samples in which either the N-terminal domain (NTD) or the C-terminal domain (CTD) is uniformly 15N,13C-labeled. Segmental labeling is achieved by trans-splicing, using the DnaE split intein. Comparisons of two-dimensional solid state NMR spectra of fully labeled and segmentally labeled tubular CA assemblies show substantial improvements in spectral resolution. The molecular structure of HIV-1 assemblies is not significantly perturbed by the single Ser-to-Cys substitution that we introduce between NTD and CTD segments, as required for trans-splicing.

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Acknowledgements

This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases and the Intramural AIDS Targeted Antiviral Program of the National Institutes of Health. We thank Dr. Marvin J. Bayro for generous assistance and helpful advice regarding HIV-1 CA assembly conditions, electron microscopy, and solid state NMR spectroscopy. We thank John R. Lloyd for performing mass spectrometry of our ligated products.

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  1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA

    Sebanti Gupta & Robert Tycko

  2. National Institutes of Health, Building 5, Room 409, Bethesda, MD, 20892-0520, USA

    Robert Tycko

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Correspondence to Robert Tycko.

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Gupta, S., Tycko, R. Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR. J Biomol NMR 70, 103–114 (2018). https://doi.org/10.1007/s10858-017-0162-1

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